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One of the most important questions in tumor biology is the understanding of the mechanisms responsible for the resistance of cancer cells to radiotherapy. In the present study, the possible role played by cell-cell interactions in determining the response of tumor cells to ionizing radiation was investigated. HT-29 colon adenocarcinoma spheroids were irradiated with a dose of 15 Gy in two different stages of growth characterized by diverse degrees of compaction: loosely organized spheroids (early spheroids) and compacted spheroids (late spheroids). Morphological analyses demonstrated that late spheroids were less damaged than early spheroids. Moreover, analyses of the cell cycle and cell death showed that ionizing radiation induced necrosis in early spheroids and apoptosis in late ones. From these results it can be concluded that late, compacted spheroids are more resistant to ionizing radiation than early, loose spheroids. In order to understand the mechanisms regulating this compaction-induced resistance of late spheroids, E-cadherin/ ß-catenin complex expression and distribution were analyzed. In late spheroids, E-cadherin/ß-catenin complexes were shown to be tethered to the cytoskeleton, and since this organization has been demonstrated to strengthen cell-cell adhesion in other systems, it can be postulated that the same is true in HT-29 spheroids. In conclusion, it can be hypothesized that compaction of HT-29 spheroids is mediated by the reorganization of E-cadherin/ß-catenin complexes on the plasma membrane and that this compaction may be responsible for the increase in resistance of HT-29 spheroids to ionizing radiation. Introduction In order to better comprehend tumor cell biology, it is necessary to utilize in vitro models that are able to represent in vivo tumors as precisely as possible. Conventional monolayer cultures used in cancer research are not completely representative of the complex microenvironmental conditions and the intricate cell-cell interactions found in solid tumors. In fact, it appears evident that the bi-dimensional organization of cells in monolayer is completely different from the threedimensional structure of solid tumors. Moreover, the homogeneous exposure to nutrients and oxygen typical of cells grown in monolayer is not representative of the heterogeneous distribution of these substances in in vivo tumors. In an attempt to overcome the experimental restrictions of monolayer cultures, in the early 1970s three-dimensional multicellular spheroids were developed. The three-dimensional organization of cells in spheroids mimics quite faithfully the intercellular interactions and the close communications between cells in solid tumors. Furthermore, this particular organization results in the development of quiescent and hypoxic (sometimes necrotic) cells in the inner zones of spheroids similar to those found in tumors. The closer resemblance of spheroids to tumors is also illustrated by the fact that multicellular spheroids respond to antineoplastic stimuli in a different manner than monolayers (1,2). In particular, it has been shown that spheroids are more resistant to chemical (antitumor drugs) and physical (ionizing radiation) agents than monolayers and that this resistance is comparable to the one found in in vivo tumors (3). Therefore, it can be theorized that studying this three-dimensional model can help elucidate the physiological, cellular and molecular mechanisms of resistance in cancer, one of the most important questions in tumor biology. Among the various hypotheses that have been formulated in order to explain the different response of monolayer cells and spheroids to stressing agents, the role of cell-cell interaction and cell communication are considered particularly important. It has been suggested that cell-cell contact could supply survival and protective advantages in cells grown as threedimensional spheroids (4). Although the molecular mechanisms of this phenomenon are not well understood, it appears evident INTERNATIONAL JOURNAL OF ONCOLOGY 28: 111-118, 2006 111 Increased cell compaction can augment the resistance of HT-29 human colon adenocarcinoma spheroids to ionizing radiation ANTONELLA FERRANTE1, GABRIELLA RAINALDI1,2, PAOLA INDOVINA1, PIETRO LUIGI INDOVINA2,3 and MARIA TERESA SANTINI1,2 1Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome; 2Istituto Nazionale per la Fisica della Materia, Unità di Napoli, 3Dipartimento di Scienze Fisiche, Università di Napoli ‘Federico II’, Complesso Universitario Monte S. Angelo, Via Cinthia, I-80126 Naples, Italy Received June 8, 2005; Accepted July 9, 2005 _________________________________________ Correspondence to: Dr Maria Teresa Santini, Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy E-mail: [email protected]